© 2017. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction

in any medium provided that the original work is properly attributed.

Long-lasting memory deficits in mice withdrawn from cocaine are concomitant to

neuroadaptations in hippocampal basal activity, GABAergic interneurons and adult

neurogenesis.

David Ladrón de Guevara-Miranda1#

, Carmelo Millón2#

, Cristina Rosell-Valle1, Mercedes Pérez-

Fernández3, Michele Missiroli

3, Antonia Serrano

4, Francisco J. Pavón

4, Fernando Rodríguez de

Fonseca4, Magdalena Martínez-Losa

3, Manuel Álvarez-Dolado

3*, Luis J. Santín

1*, Estela Castilla-

Ortega1,4

*

1Departamento de Psicobiología y Metodología de las Ciencias del Comportamiento, Instituto de Investigación

Biomédica de Málaga (IBIMA), Facultad de Psicología, Universidad de Málaga, Spain.

2Departamento de Fisiología, Instituto de Investigación Biomédica de Málaga (IBIMA), Facultad de Medicina,

Universidad de Málaga, Spain.

3Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Sevilla, Spain

4Unidad de Gestión Clínica de Salud Mental, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital

Regional Universitario de Málaga, Spain.

#These authors contributed equally to this work.

*Correspondence to:

Manuel Álvarez-Dolado. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Av.

Americo Vespucio s/n, 41092 Sevilla, Spain. E-mail: manuel.alvarez@cabimer.es

Luis J. Santín. Departamento de Psicobiología y Metodología de las CC, Facultad de Psicología, Universidad de

Málaga, Campus de Teatinos S/N, 29071 Málaga, Spain. E-mail: luis@uma.es

Estela Castilla Ortega. Unidad de Gestión Clínica de Salud Mental, Instituto de Investigación Biomédica de

Málaga (IBIMA), Hospital Regional Universitario de Málaga, Avenida Carlos Haya 82, 29010 Málaga, Spain.

E-mail: estela.castilla@ibima.eu

Keywords

Anxiety; early-immediate gene c-Fos; parvalbumin PV; neuropeptide Y NPY; cell proliferation;

behavior-induced neuroplasticity

Summary statement

Hippocampal functional activity and neuroplasticity are altered in mice long withdrawn from cocaine,

concomitantly to memory deficits.

Results emphasize the hippocampal role in the persistent cognitive manifestations of cocaine

withdrawal.

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.026682Access the most recent version at DMM Advance Online Articles. Posted 30 January 2017 as doi: 10.1242/dmm.026682http://dmm.biologists.org/lookup/doi/10.1242/dmm.026682Access the most recent version at

First posted online on 30 January 2017 as 10.1242/dmm.026682

Abstract

The cocaine addiction disorder is notably aggravated by concomitant cognitive and emotional

pathology that impedes recovery. We studied whether a persistent cognitive/emotional dysregulation

in mice withdrawn from cocaine holds a neurobiological correlate within the hippocampus, a limbic

region with a key role in anxiety and memory but that has been scarcely investigated in cocaine

addiction research. Mice were submitted to a chronic cocaine (20 mg/kg/day for 12 days) or vehicle

treatment followed by 44 drug-free days. Some mice were then assessed on a battery of emotional

(elevated plus-maze, light/dark box, open field, forced swimming) and cognitive (object and place

recognition memory, cocaine-induced conditioned place preference, continuous spontaneous

alternation) behavioral tests, while other mice remained in their home-cage. Relevant hippocampal

features [basal c-Fos activity, GABA+, parvalbumin (PV)+ and neuropeptide Y (NPY)+ interneurons,

and adult neurogenesis (cell proliferation and immature neurons)] were immunohistochemically

assessed 73 days after the chronic cocaine or vehicle protocol. The cocaine-withdrawn mice showed

no remarkable exploratory or emotional alterations but were consistently impaired in all the cognitive

tasks. All the cocaine-withdrawn groups, independently of whether they were submitted to behavioral

assessment or not, showed enhanced basal c-Fos expression and increased number of GABA+ cells

in the dentate gyrus. Moreover, the cocaine-withdrawn mice previously submitted to behavioral

training displayed a blunted experience-dependent regulation of the dentate gyrus’ PV+ and NPY+

neurons, and adult hippocampal neurogenesis. Results highlight the importance of hippocampal

neuroplasticity for the ingrained cognitive deficits present during chronic cocaine withdrawal.

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INTRODUCTION

The use of illicit drugs is one of the most serious health problems in the Western world, and cocaine is

the most widely used psychostimulant drug (EMCDDA, 2015; UNODC, 2015). Cocaine abuse usually

carries an important socioeconomic burden with both physical and mental health dysfunctions,

including the engagement in a cocaine addiction disorder when casual users become dependent on

this substance (Lopez-Quintero et al., 2011). A growing body of literature supports the premise that

persistent emotional and cognitive symptoms contribute to the chronic and relapsing nature of cocaine

addiction. At the emotional level, cocaine withdrawal is often associated with negative affect involving

a high anxiety state and intense desire for the drug (‘craving’) that urges the individual to resume drug

intake (Koob and Zorrilla, 2010). Moreover, there is an elevated incidence of mood and anxiety

disorders among cocaine addicts (Araos et al., 2015; Araos et al., 2014; Pedraz et al., 2015a; Pedraz

et al., 2015b) and such psychiatric comorbidity yields a worse prognosis (Siqueland et al., 2002). At a

different level, cocaine dependent individuals show broad cognitive deficits [including attention,

working memory, reference memory, behavioral inhibition or cognitive flexibility (Spronk et al., 2013;

Vonmoos et al., 2013; 2014)] that are therapeutically relevant as they predict relapse and low

treatment retention (Aharonovich et al., 2006; Fox et al., 2009; Teichner et al., 2002). These affective

and cognitive symptoms in cocaine addicts are, at least in part, caused by the repeated cocaine use

(Vonmoos et al., 2014), and pre-clinical studies have evidenced that cocaine exposure and withdrawal

induce both emotional (Perrine et al., 2008; Sarnyai et al., 1995) and cognitive alterations (Briand et

al., 2008; Krueger et al., 2009; Mendez et al., 2008) in rodents.

The hippocampal region is emerging as a strong candidate that significantly contributes to the

cocaine-induced emotional and cognitive symptoms (Castilla-Ortega et al., 2016b). This limbic

structure has a long established role to regulate anxiety and depression-like responses, as well as key

cognitive functions such as working and reference memory (Bannerman et al., 2004; Walf and Frye,

2006). As revealed by post-mortem samples, the hippocampus from cocaine addicts shows a notable

dysregulation in the expression of genes involved in glutamatergic and GABAergic transmission

(Enoch et al., 2014; Enoch et al., 2012) as well as in cellular plasticity and function (Mash et al., 2007;

Zhou et al., 2011). Moreover, the hippocampus is integrated into the ‘addiction brain circuit’ with the

main reward areas, establishing reciprocal functional connections that are sculpted by cocaine

exposure (Castilla-Ortega et al., 2016b). Resting-state functional neuroimaging studies in cocaine

addicts reveal a whole-brain functional connectivity reduction where the hippocampus stands out as

one of the brain structures most evidently disconnected from other regions (Castilla-Ortega et al.,

2016b; Ding and Lee, 2013). In experimental conditions where the cocaine dependent individuals are

exposed to cocaine associated-cues, hippocampal activity is then greatly increased and correlated

whith craving (Castilla-Ortega et al., 2016b), while higher hippocampal activation in resting conditions

predicts the likelihood to relapse in cocaine use (Adinoff et al., 2015). Considering that poorer

cognitive performance –affecting hippocampal-dependent functions such as spatial ability and verbal

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memory- predicts treatment dropout and further cocaine intake in cocaine addicts receiving treatment

(Aharonovich et al., 2006; Fox et al., 2009; Teichner et al., 2002), it is possible for the hippocampus to

hold a common neurobiological substrate that underlies both the impaired cognition and the relapse

outcomes in cocaine addicts, thus being a relevant therapeutic objective. Nevertheless, the

hippocampus has received relatively scarce attention in cocaine addiction research as it is not among

the classical addiction-related brain areas [e.g.(Everitt, 2014; Everitt et al., 2008)].

Hippocampal activity and function are tightly regulated by neuroplastic and neurogenic processes in

the dentate gyrus (DG), a region that is the main input for the hippocampus and highly responsive to

the environmental demands (Castilla-Ortega et al., 2011). In fact, the DG is one of the few neurogenic

niches in the adult brain. Adult hippocampal neurogenesis (AHN) is required for normal hippocampal

function, since its ablation results in aberrant emotionality (Revest et al., 2009) and impaired cognition

(Castilla-Ortega et al., 2011; Deng et al., 2010; Leuner et al., 2006). In contrast, the increment of AHN

potentiates memory and alleviates emotional dysregulation (Castilla-Ortega et al., 2011; Deng et al.,

2010; Leuner et al., 2006). Importantly, the adult-born DG neurons are modulated by a number of

stimuli that may upregulate (e.g. hippocampal-dependent learning activities, physical exercise) or

reduce (e.g. stressful experiences) their proliferation, maturation and/or survival, with subsequent

implications for behavior (Castilla-Ortega et al., 2011; Deng et al., 2010; Leuner et al., 2006). Another

key neuron population that modulates hippocampal function are the gamma-amino butyric acid

(GABA)-ergic interneurons, which constitute a ~10% of the total hippocampal neurons, and include

multiple inhibitory neuron sub-types according to their neurochemical identity (Freund and Buzsaki,

1996; Vizi and Kiss, 1998). In particular, those expressing parvalbumin (PV) or neuropeptide Y (NPY)

have demonstrated a role in both anxiety and memory. Thus, increment in the number of these

interneurons usually correlates with anxiolysis and a boosted cognitive performance, whereas their

loss leads to detrimental behavioral effects and is associated with several pathological conditions [PV:

(Boley et al., 2014; Brady and Mufson, 1997; Fuchs et al., 2007; Korotkova et al., 2010; Megahed et

al., 2014; Morellini et al., 2010; Quarta et al., 2015; Zou et al., 2016); NPY: (Beck and Pourie, 2013;

Christiansen et al., 2014; de Lanerolle et al., 1989; Megahed et al., 2014)]. These neuronal

populations also undergo experience-dependent modulation after exercise, environmental enrichment,

stress and learning (Arida et al., 2004; Bjornebekk et al., 2006; 2007; Czeh et al., 2005; Donato et al.,

2013; Filipovic et al., 2013; Iuvone et al., 1996; Nguyen et al., 2013; Sergeyev et al., 2005; Suzuki et

al., 2014). Interestingly, the NPY stimulates AHN (Decressac et al., 2011; Howell et al., 2005) and the

PV+ interneurons are involved in early key phases of the AHN process (Song et al., 2013). Therefore,

both the AHN, PV and NPY-expressing neurons are interrelated and significantly contribute to the

neuroplasticity that regulates hippocampal function and its adaptability to the environmental demands.

In the field of cocaine research, it is known that rodents with reduced AHN engage in more cocaine

seeking and taking behaviors (Castilla-Ortega et al., 2016a; Deschaux et al., 2014; Noonan et al.,

2010) and that cocaine exposure usually reduces AHN (Blanco-Calvo et al., 2014; Castilla-Ortega et

al., 2016b). However, young hippocampal neurons and proliferating cells are apparently normalized

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shortly after cocaine cessation, which complicates the establishment of a strong link between an

impaired AHN and the long-lasting cocaine-induced behavioral symptoms (Castilla-Ortega et al.,

2016b; Mackowiak et al., 2005; Xie et al., 2010; Yamaguchi et al., 2005). Nevertheless, it remains to

be investigated whether AHN normally responds to the environmental stimuli during cocaine

withdrawal. On the other hand, a potential effect of cocaine on the hippocampal GABAergic

interneurons, including the PV+ and NPY+ populations, has been largely unexplored. The present

study aims to investigate whether emotional and cognitive symptoms and their associated

hippocampal features may persist in mice long withdrawn from cocaine, with special emphasis on the

neurogenic DG. Cocaine’s effects on basal hippocampal activity (assessed by early-immediate gene

c-fos expression); GABA+, PV+ and NPY+ hippocampal neurons; and AHN (proliferating cells and

young DG neurons) were analysed by immunohistochemistry, both in undisturbed mice and in mice

previously submitted to behavioral training in a battery of emotional and memory tests. In this way, we

will research how summiting mice to a cognitively demanding environment modulates GABAergic

interneurons and AHN in the cocaine-treated mice.

MATERIALS AND METHODS

Animals

Forty-four male C57BL/6J mice acquired from Janvier Labs (Le Genest-Saint-Isle, France) arrived the

animal facility at eight weeks of age and underwent one week of acclimatization before the

experiments started. Mice were housed in groups of 3-4 mice on a 12 h light/dark cycle in standard

cages containing shredded paper as nesting material, water and food provided ad libitum. All

experiments were performed in accordance with the European (Directive 2010/63/UE) and Spanish

(Real Decreto 53/2013, Ley 32/2007 and 9/2003) regulations of animal research. The experimental

protocols were approved by the research ethics committee of the University of Málaga (code: CEUMA

8-2014-A and 19-02-15-197).

Cocaine treatment and experimental conditions

Mice were randomly assigned to a cocaine (‘COC’, n = 22) or a vehicle (‘VEH’, n = 22) condition. The

‘COC’ mice received a chronic cocaine treatment consisting of a daily intraperitoneal (i.p.) 20 mg/kg

dose of cocaine (Sigma, St. Louis, USA; diluted in 10 ml/kg volume of saline –0.9 % NaCl-) during 12

consecutive days; while the VEH mice received an equivalent i.p. saline volume. After the last cocaine

or saline dose, all mice remained undisturbed for 44 days (Fig. 1). From days 45 to 62, both groups

were split in the ‘-Behav’ mice (Fig. 1a; COC-Behav, n = 11; VEH-Behav, n = 10) that were submitted

to a behavioral test battery to evaluate hippocampal dependent behavior, including training in a

cocaine-induced conditioned place preference (CPP) paradigm; and the ‘-Control’ mice (Fig. 1b; COC-

Control, n = 5; VEH-Control, n = 6) that were not exposed to any behavioral training but received

home-cage cocaine injections (2.5 mg/kg/day on days 52-55; 10 mg/kg/day on days 57-60), as control

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for the drug administration that -Behav groups received during their CPP training. Therefore, the –

Behav and the –Control groups only differed in their exposure to training stimulation, so their

comparison allows to determining the impact of behavior on the histological parameters. The

remaining mice (‘-Basal’) were included to assess the effects of the initial VEH- or COC- treatment in

conditions of no further drug or behavioral stimulation. Thus, -Basal mice were exposed to home-cage

vehicle injections during days 52-55 and 57-60 (Fig. 1c; VEH-Basal, n = 6; COC-Basal, n = 6). The

experimental groups submitted to behavioral assessment used an increased sample size because the

behavioral data are usually more variable than the histological results.

Behavioral Assessment

The behavioral tests (Fig. 1a) were performed on the basis of previously published protocols (Albasser

et al., 2010; Castilla-Ortega et al., 2016a; Hughes, 2004; Ladron de Guevara-Miranda et al., 2016;

Santin et al., 2009) as detailed in the Supplementary methods. Briefly, for an evaluation of

exploratory and anxiety-like behavior mice underwent one session in the elevated plus maze (day 45

after the last vehicle or cocaine dose), in the light/dark box (day 46) and in the open field (day 47)

tests. Cognitive function was subsequently assessed through a novel object recognition task (Fig. 3a)

that measured memory for a familiar object (day 48) calculated as an ‘object memory ratio’ [(time

exploring the novel object – time exploring the familiar object)/ total time exploring both objects] and

memory of a familiar place (day 49) calculated as a ‘place memory ratio’ [(time exploring the displaced

object – time exploring the static object)/ total time exploring both objects] (Supplementary methods).

According to these ratios, a successful object or place memory would be indicated by a positive ratio

score that is significantly different from zero (Albasser et al., 2010). Finally, depression-like behavior

was evaluated in the forced swimming test (day 50). The rationale underlying this test order was to

assess exploration and anxiety-like behavior first, when animals are less habituated to the

environment so it would elicit more novelty and aversion; while a high stressful task such as the forced

swimming test was administered last to avoid its potential impact on later behavioral measures.

Cocaine-induced locomotion and the acquisition of drug-contextual associations were subsequently

evaluated in a CPP paradigm. In one habituation session (day 51), the –Behav mice freely explored a

CPP apparatus that consisted of two compartments distinguishable by the contextual cues decorating

their walls and communicated by a clear corridor. The time spent in each compartment was recorded

and the compartment least preferred by a mouse was paired with a cocaine injection in the

conditioning phase, while the other compartment was paired with a saline injection. To study the

effects of both a low and a high cocaine dose, conditioning was first carried out with a dose of 2.5

mg/kg/day (days 52-55) of cocaine followed by a test session (Test 1; day 56); and then with a dose of

10 mg/kg/day of cocaine (days 57-60), followed by a second test session (Test 2; day 61). Test

sessions consisted of free exploration of the CPP apparatus after saline administration. A ‘CPP ratio’

[(seconds spent in the cocaine-paired compartment - seconds spent in the saline-paired compartment)

/ total seconds spent in both compartments]*100 was calculated, so the preference for the cocaine-

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paired compartment over the saline-paired one would be indicated by a positive CPP ratio,

significantly different from zero (Castilla-Ortega et al., 2016a; Ladron de Guevara-Miranda et al., 2016;

Poltyrev and Yaka, 2013).

The continuous spontaneous alternation in the Y-maze was evaluated at the end of the testing (day

62). An ‘alternation score’ [(number of spontaneous alternations)/(total number of arm entries – 2)]

(Hughes, 2004) was calculated for the first 30 possible alternations (i.e. 32 arm entries) performed.

Immunohistochemistry and cell quantification

On day 73, mice from all experimental conditions were deeply anesthetized for transcardial perfusion

with 0.1 M phosphate-buffered saline pH 7.4 (PBS). Their brains were dissected out and cut through

midline. The left hemisphere was arbitrarily chosen for the histological study. It was postfixed for 48 h

in 4 % paraformaldehyde in PBS and cut into coronal vibratome sections (50 µm) from -1.06 to -3.08

mm from bregma (Paxinos and Franklin, 2001) that include the hippocampal area. For free-floating

immunohistochemistry, sections first received a heat-induced epitope retrieval (EnVision Flex high pH

solution; Dako, Glostrup, Denmark) followed by an endogenous peroxidase blocking (80 % PBS, 10 %

methanol and 10 % hydrogen peroxidase) for 30 min in the dark, and they were incubated overnight

with the corresponding primary antibody. A mouse anti-c-Fos antibody (1:500, Santa Cruz

Biotechnology, Santa Cruz, USA; sc-271243; Lot # 10413; -which is also reactive for the c-Fos

functional homologs Fos B, Fra-1 and Fra-) was used for detection of basal neuronal activity; while the

GABAergic interneuron populations were assessed by rabbit anti-PV (1:500; Swant, Marly,

Switzerland, PV-28; Lot # 5.10), rabbit anti-NPY (1:500, Sigma, N9528; Lot # R40829) and rabbit anti-

GABA (1:500, Sigma, A2052; Lot # 095K4830) antibodies. The AHN-related markers used were a

mouse anti-Proliferating Cell Nuclear Antigen (PCNA; 1:1000; Sigma, P8825; Lot # 014M4836) to

label cells undergoing proliferation in the dentate gyrus, and goat anti-doublecortin (DCX; 1:200; Santa

Cruz, sc-8066; Lot # A1211) for immature neurons up to 3-4 weeks of age (Brown et al., 2003). All

antibodies were diluted in PBS, 0.5 % Triton X-100 and 2.5 % donkey serum. On the second day,

sections were incubated for 90 min in a biotin-conjugated secondary antibody (rabbit anti-mouse,

rabbit anti-goat or swine anti-rabbit as appropriate; Dako; diluted 1:800) and for 1 h in peroxidase-

conjugated extravidin (Sigma, 1:1000 in PBS) solution in the dark. The staining solution contained

diaminobenzidine (DAB) as the chromogen (proportion: 0.1 ml of DAB previously diluted at 5 % in

distilled water, 10 µl hydrogen peroxidase and 10 ml PBS) and nickel chloride (NiCl2, Sigma) was

added to intensify c-Fos labelling (0.004 gr per 10 ml of staining solution). Each step was followed by

PBS rinses. Negative controls in which the primary antibody was omitted resulted in absent staining.

Additional controls for antibody specificity and selectivity (using western blot, ELISA, knockout

mice,…) as well as scientific literature employing these antibodies are available from the

manufacturers, either in their website or upon request.

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Markers were quantified separately in the supragranular (SupraG) and infragranular (InfraG) cell

layers of the DG in all mice, while other DG regions and the cornu ammonis (CA) were further

explored in the –Behav mice groups. Importantly, it has been recently emphasized that the SupraG

and InfraG blades should be considered as different functional and morphological divisions within the

DG (Gallitano et al., 2016; Jinno, 2011). Cell counting was carried out following our previously

reported methods (Castilla-Ortega et al., 2016a; Castilla-Ortega et al., 2013; Castilla-Ortega et al.,

2014). Cells were quantified out in 1 of every 8 hippocampal sections for each marker in high

magnification photographs (4080 x 3072 pixels, using a 10x lens –for the DG- or a 4x lens –for the CA)

captured with an Olympus BX51 microscope equipped with an Olympus DP70 digital camera

(Olympus, Glostrup, Denmark). The boundaries of the different hippocampal regions were defined

according to anatomical criteria (DG: molecular, SupraG, InfraG and polymorphic layers; CA1: stratum

oriens, pyramidal, radiatum and lacunosum; CA3: stratum oriens, pyramidal and radiatum) (Paxinos

and Franklin, 2001). Using the software ImageJ (US National Institute of Health, Maryland, USA), the

area of each region of interest was drawn, measured, and the positive cells within the region were

manually counted. Data of each animal was expressed as the average number of positive cells per

unit area (mm2). In addition, the DCX+ cells were classified into two cell types: ‘Type 1’: these were

cells with absent or short dendritic processes (a morphology that usually corresponds to the most

immature DCX+ cells) or ‘Type 2’: which were mature-like cells with at least one prominent apical

dendrite penetrating the granule cell layer (Castilla-Ortega et al., 2016a; Castilla-Ortega et al., 2014;

Plumpe et al., 2006).

Statistical analysis

Comparisons among groups were carried out with Student’s t-tests; or with repeated-measures

analysis of variance (ANOVA) followed by post-hoc Fisher’s least significant difference (LSD) analysis

for between-groups comparisons when appropriate (Tybout et al., 2001). A one-sample t-test was

used to compare means to a single measure. Significance was considered at p ≤ 0.05. Only significant

comparisons are reported.

To simplify and reduce the cognitive-related behavioral data, a principal component factorial analysis

was performed as described in the Supplementary methods. In addition, to confirm whether

behavioral training differently modulated hippocampal neuroplasticity in the cocaine withdrawn mice,

expression data from each histological marker in the -Behav mice was transformed to its relative

change (Δ) from the respective -Control group (Supplementary methods).

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RESULTS

Cocaine-withdrawn mice show minimal emotional alterations in the absence of exploratory changes

In order to analyse anxiety-like behavior we performed open field, elevated plus maze and dark/bright

field tests. Groups were similar in most relevant measures of anxiety in these tests, such as total time

spent in the open arms of the elevated plus maze (Fig. 2a); the number of entries and the total time

spent in the bright compartment of a light/dark box (Fig. 2b); or exploration of the centre area in the

open field (Fig. 2c). The only significant change was a reduced number of entries in the plus maze

open arms in the COC-Behav mice (t(19)= -2.105, p = 0.049), in the absence of locomotor deficits

(Fig. 2a). On the other hand, the forced swimming test did not reveal differences in depression-like

behavior assessed as the latency for first immobility and total immobility time (Fig. 2d), nor in the time

the mice struggled in the water (data not shown).

Chronic cocaine exposure induces persistent cognitive impairment

Despite the absence of notable alterations in their emotional and exploratory behavior, the cocaine-

withdrawn mice showed a consistent memory deficit. In contrast to the VEH-Behav group, the COC-

Behav mice were impaired to discriminate both a novel (t(19) = 3.933, p = 0.001) and a displaced

object (t(19) = 2.450, p = 0.024), performing by chance in both tasks (Fig. 3b). It is important to note

that mice of both treatments spent a similar amount of time exploring objects across trials (Fig. 3a),

ruling out a reduced motivation for object exploration.

During the CPP training, the locomotor activity induced by cocaine administration was analysed by a

repeated measures ANOVA [‘treatment’ (VEH or COC) x ‘cocaine dose’ (2.5 or 10 mg/kg) x

‘compartment’ (saline- or cocaine-paired) x ‘session’ (1-4)], that revealed significant effects [‘treatment

x compartment’: F(1, 76) = 4.240, p = 0.043; ‘cocaine dose’: F(1, 76) = 115.940, p = 0.000;

‘compartment’: F(1, 76) = 120.53, p = 0.000; ‘dose x compartment’: F(1, 76) = 77.235, p = 0.000;

‘session x cocaine dose’: F(3, 228) = 5.500, p = 0.001; ‘session x cocaine dose x compartment’: F(3,

228) = 4.082, p = 0.008)]. The post-hoc comparisons showed an exacerbated locomotor response to

the 10 mg/kg cocaine dose in the COC-Behav mice. This hyperlocomotion was most evident in the

first exposures since the VEH-Behav mice were progressively sensitized to the locomotor effects of

the drug (LSD is shown in Fig. 3c). However, both groups showed similar locomotion after saline

administration or after the 2.5 mg/kg cocaine dose, which was insufficient to induce any stimulating

effects. Regarding the expression of the cocaine-induced conditioned response, groups were similar in

the habituation session (p > 0.05) but a repeated measures ANOVA across the Test sessions

revealed worse conditioning in the COC-Behav mice (‘treatment’: F(1, 19) = 4.736, p = 0.042;

‘session’: F(1, 19) = 7.734, p = 0.012; Fig. 3d).

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Spatial working memory was further evaluated in a Y maze. The COC-Behav mice performed by

chance and achieved fewer correct spontaneous alternations than the VEH-Behav mice (t(19) = -

3.283, p = 0.004), while no effects were found in locomotion (Fig. 3e).

Lastly, a factor analysis (Supplementary methods) extracted one unique factor in which all the

memory-related measures were included (Fig. 3f), representing mice’s cognitive performance.

Comparing mice’s factor scores determined a superior cognitive performance in the VEH-Behav group

(t(19) = 4.359, p = 0.000; Fig. 3f).

Withdrawal from chronic cocaine induces a persistently increased DG basal activity

Mice in the –Behav and –Control conditions were compared by a repeated measures ANOVA to

assess the impact of chronic cocaine withdrawal and behavioral training on the histological parameters

of both the SupraG and InfraG cell layers of the DG [‘treatment’ (VEH or COC) x ‘behavior’ (-Control or

-Behav) x ‘DG blade’ (SupraG or InfraG)].

Basal hippocampal c-Fos expression was evaluated since ‘resting’ brain activity and functional

connectivity are profoundly altered in the hippocampus of cocaine addicts (Adinoff et al., 2015; Ding

and Lee, 2013). Both the COC-Control and the COC-Behav mice displayed increased basal c-Fos

expression in the SupraG region compared to their respective VEH groups (Fig. 4a,b) (‘treatment’:

F(1, 28) = 10.899, p = 0.003; ‘DG blade’: F(1, 28) = 172.39, p = 0.000; ‘treatment x DG blade’: F(1, 28)

= 12.776, p = 0.001; LSD is shown in Fig. 4b) and there was also an effect for the behavioral training

in reducing basal c-Fos activity in the SupraG (‘DG blade x behavior’: F(1, 28) = 16.594, p = 0.000;

Fig. 4a; and analysis in Fig. 7a). Interestingly, an increased c-Fos expression in the DG was also

found in the COC-Basal mice (‘treatment’: F(1, 10) = 10.235, p = 0.010; ‘DG blade’: F(1, 10) = 261.31,

p = 0.000; LSD is shown in Fig. 4c). These results revealed a persistently increased basal c-Fos

expression in the DG of cocaine-withdrawn mice, irrespectively of their behavioral treatment.

Interestingly, this feature seemed limited to the DG, since further exploration of this marker (carried out

in the VEH-Behav and COC-Behav groups) revealed no effects of cocaine on c-Fos expression in the

pyramidal cell layers (Supplementary Table).

Cocaine-withdrawn mice show altered regulation of DG GABAergic neurons and adult hippocampal

neurogenesis

Similarly to the c-Fos result, a persistent upregulation of the GABA+ neurons was found in the DG in

all the cocaine-withdrawn groups (‘DG blade’: F(1, 28) = 11.651, p = 0.002; ‘treatment x DG blade’:

F(1, 28) = 9.830, p = 0.004; LSD is shown in Fig. 5a,d; this effect was replicated in the -Basal mice

shown in Supplementary Figure 1). Behavioral training increased the number of GABA+ neurons in

the DG, but this enhancement was not modulated by cocaine withdrawal, as it was similar in both

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VEH- and COC-treated mice (‘behavior’: F(1, 28) = 11.651, p = 0.002 in Fig. 5a,d; and analysis in

Fig.7b).

On the other hand, withdrawal from chronic cocaine did not induce long-lasting alterations in the DG’s

PV+ and NPY+ interneurons populations (Fig. 5b,c) or in the AHN-related markers (proliferating

PCNA+ cells and DCX+ immature neurons; Fig. 6), when these were assessed under ‘resting’

conditions. It was observed that the numbers were similar in the VEH-Control and the COC-Control

mice, and results in the –Basal groups did not reveal differences either (Supplementary Figure 1).

The behavioral training notably increased hippocampal plasticity by enhancing the expression of both

PV+ and NPY+ neurons (ANOVA effects for PV: ‘behavior’: F(1, 28) = 6.080, p = 0.020; ‘DG blade’:

F(1, 28) = 17.804, p = 0.000; ‘treatment x behavior x DG blade’: F(1, 28) = 4.177, p = 0.050; ANOVA

effects for NPY: ‘DG blade’: F(1, 28) = 68.400, p = 0.000; ‘DG blade x behavior’: F(1, 28) = 9.128, p =

0.005; LSD is shown in Fig. 5b,c,e,f) and by modulating AHN-related markers (ANOVA effects for

PCNA: ‘behavior’: F(1, 28) = 29.728, p = 0.000; ‘DG blade’: F(1, 28) = 10.468, p = 0.003; ‘DG blade x

treatment’: F(1, 28) = 8.758, p = 0.006; ‘treatment x behavior x DG blade’: F(1, 28) = 4.396, p = 0.045;

ANOVA effects for the % of mature-like ‘Type 2’ DCX: ‘behavior’: F(1, 28) = 9.935, p = 0.004; ‘DG

blade’: F(1, 28) = 111.00, p = 0.000; ‘DG blade x treatment’: F(1, 28) = 20.007, p = 0.000; ‘treatment x

behavior x DG blade’: F(1, 28) = 5.128, p = 0.031; LSD is shown in Fig. 6a,c,d,e). Nonetheless, post-

hoc comparisons revealed differences between the VEH-Behav and the COC-Behav mice suggesting

that they underwent a different neuroplastic modulation after behavior in certain DG blades (Fig. 5, 6).

Therefore, the impact of the behavioral training on the DG during cocaine withdrawal was further

explored by analysing the relative change found in the –Behav mice groups from their respective -

Control group (Supplementary methods). Results confirmed that the COC-Behav mice showed a

blunted increase of both PV+ and NPY+ cells in the SupraG region (ANOVA effects: for PV: ‘treatment

x DG blade’: F(1, 19) = 12.114, p = 0.003; ANOVA effects for NPY: ‘treatment’: F(1, 19) = 10.176, p =

0.005; ‘DG blade’: F(1, 19) = 39.062, p = 0.000; ‘treatment x DG blade’: F(1, 19) = 8.073, p = 0.010;

LSD is shown in Fig. 7c,d). In addition, the COC-Behav mice dysregulated their expression of PCNA

(‘DG blade’: F(1, 19) = 5.064, p = 0.036; ‘treatment x DG blade’: F(1, 19) = 11.692, p = 0.003; Fig. 7e)

and DCX in the InfraG layer. They showed a blunted cell proliferation that did not affect the total

number of young neurons. However, there was a reduction in the frequency of ‘Type 2’ mature-like

morphology cells (‘treatment x DG blade’: F(1, 19) = 5.474, p = 0.030; LSD is shown in Fig. 7f,g).

Finally, it is worth mentioning that the VEH–Behav and COCA–Behav groups showed no remarkable

differences in their GABAergic neuron populations assessed in the CA1 and CA3 hippocampal regions

(Supplementary Table) or in the turnover of the adult-born cells assessed by the amount of apoptotic

nuclei in the granular and subgranular zones (Supplementary Figure 2).

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DISCUSSION

This study describes long-lasting memory deficits in mice withdrawn from cocaine, that show

persistent neuroadaptations in the hippocampal DG concerning increased basal neuronal activity as

well as an altered regulation of the GABAergic neurons and AHN after a behavioral training. This pre-

clinical evidence will help to characterize the neurobiological basis of the hippocampal-dependent

emotional and cognitive alterations ocurring during cocaine withdrawal.

Mice were submitted to a chronic cocaine treatment for 12 days and then to 44 days of a drug-free

period before the behavioral assessment started. The cocaine-withdrawn mice displayed normal

exploratory behavior across testing, and their emotional alterations were subtle since only the plus

maze test (that probably elicited the most anxiogenic situation considering that it was performed in the

first place, when mice were less habituated to the testing procedure) revealed a tendency to increased

anxiety in the cocaine-treated animals. These results apparently disagree with previous works

indicating that both acute and chronic cocaine exposure have clear anxiogenic properties (Paine et al.,

2002; Yang et al., 1992) and that drug withdrawal is characterized by a high anxiety state that may

course with defenseless behaviors in rodents (Alves et al., 2014; El Hage et al., 2012; Erb et al., 2006;

Harris and Aston-Jones, 1993; Perrine et al., 2008; Santucci and Rosario, 2010; Sarnyai et al., 1995;

Valzachi et al., 2013). Nonetheless, it should be highlighted that the aforementioned experiments used

cocaine withdrawal periods significantly shorter (24 h to 28 days) than our experimental protocol (44

days), suggesting that anxiety may gradually decline after prolonged cocaine abstinence.

On the other hand, the cocaine-withdrawn mice displayed notable cognitive deficits as reflected by

impaired reference memory (24 h recognition of familiar objects and locations), impaired acquisition of

drug-contextual associations (CPP test) and impaired spatial working memory (continuous

spontaneous alternation in the Y maze). It should be noted that this study does not provide direct

evidence on the degree of hippocampal participation in the aforementioned tasks. In this regard, the

hippocampal involvement in object recognition memory is controversial, as it may depend on several

aspects in the testing protocol [such as complexity of the testing environment (Denny et al., 2012) or

the temporal delay between the sample and test sessions (Cohen and Stackman, 2015)].

Nevertheless, literature reports a clear role of the hippocampus for both the place recognition memory

(Barker and Warburton, 2011; Broadbent et al., 2004) and the acquisition and processing of cocaine-

stimuli associations in the CPP paradigm (Castilla-Ortega et al., 2016b; Hernandez-Rabaza et al.,

2008). Thus, our results strongly support a defective hippocampal-dependent memory in the cocaine-

withdrawn mice.

Hippocampal-dependent memory impairment has been reported in rodents withdrawn from chronic

cocaine (Briand et al., 2008; Burke et al., 2006; Krueger et al., 2009) even after a long withdrawal

period of three months (Mendez et al., 2008). A novelty of this study is that multiple tasks were

performed to evaluate both emotion and cognition. The results point out that the cocaine-induced

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cognitive impairment is independent of the emotional symptoms, and this alteration may persist over

time even after recovery at the emotional level. Also, because emotion and memory are supported by

different neurobiological mechanisms and are dissociated within the hippocampus (Bannerman et al.,

2004; Bertoglio et al., 2006), it is possible to explain a different performance in these two domains.

Hippocampal histological markers were evaluated at day 73 of cocaine withdrawal. A main finding is

that the cocaine-withdrawn mice, regardless of whether they were submitted to behavioral training or

not, displayed an enhanced basal c-Fos activity in the hippocampus, specifically located in the DG.

This shows persistent neuroadaptations increasing DG basal excitatory tone during cocaine

withdrawal that likely impairs further response of the hippocampus to environmental/behavioral

demands, since the hippocampal neuronal activity supports the encoding and consolidation of stimuli

into memories (Kheirbek et al., 2013). The enhanced basal c-Fos activity was paralleled to an up-

regulation of the GABA+ neurons in the DG region, suggesting compensatory mechanisms attempting

to enhance the inhibition. However, the expression of markers for specific GABAergic interneuron

populations, such as PV and NPY, was not increased in the cocaine-withdrawn animals. A possible

explanation for this discrepancy is that the enhanced GABA+ cell population in the cocaine-withdrawn

DG not only may comprise interneuron subtypes such as the ones expressing PV and NPY, but

excitatory granular projection neurons that modified their neurochemical content (Kahn et al., 2005). It

has been widely reported that glutamatergic DG neurons constitutively contain both GABA and its

synthesis enzymes, and they may rapidly change to a GABAergic phenotype in pathological conditions

of hippocampal hyper-excitation (Makiura et al., 1999; Ramirez and Gutierrez, 2001; 2003; Sloviter et

al., 1996), as may happen after cocaine withdrawal. Interestingly, an increment of GABA-synthesizing

neurons in the DG has been described in mice abstinent from other dependence-inducing drugs

(morphine) (Kahn et al., 2005). To the best of our knowledge, few previous studies have investigated

the impact of cocaine on the hippocampal GABAergic neurons, and none tested the animals after long

withdrawal periods. The available evidence (Goodman and Sloviter, 1993) is in concordance with our

result as it showed that a repeated high cocaine dosage does not apparently induce neurotoxicity, nor

alters basal expression of GABAergic interneuron populations markers in the hippocampus [numbers

of PV+, calretinin+ and somatostatin+ neurons were unchanged; changes were found in NPY-

immunoreactivity but these were elicited by cocaine-induced seizures and not by cocaine

administration itself].

Thus, under non-stimulating conditions, the cocaine-withdrawn mice showed no evident alterations in

the expression of PV and NPY in the DG; and their AHN-related markers (cell proliferation and DCX+

neurons) were also normal. In contrast, these populations were significantly altered in the cocaine-

withdrawn mice submitted to behavioral training. The behavioral training used in this study included a

number of hippocampal-relevant stimuli, such as exposure to novel contexts and objects,

locomotion/exercise opportunities, mild anxiogenic experiences (i.e. unknown environments and

inescapable situations), and engagement in learning activities. Thus, while it is not possible to link a

particular stimulus or behavior to its corresponding neurobiological correlate, the whole behavioral

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training protocol could be understood as an ‘enriched’ or hippocampal-demanding environment that

may have an impact on the hippocampus. Both AHN and the hippocampal PV+ and NPY+ populations

have been demonstrated to increase their expression in response to exercise (Arida et al., 2004;

Bjornebekk et al., 2006; Nguyen et al., 2013; van Praag et al., 1999), environmental enrichment

(Bjornebekk et al., 2007; Iuvone et al., 1996; Kempermann et al., 1997; Suzuki et al., 2014) and/or

hippocampal-dependent learning (Donato et al., 2013; Gould et al., 1999). Accordingly, our results

showed that normal mice submitted to behavioral training potentiated DG’s inhibitory mechanisms, as

reflected by a reduction in basal c-Fos activity with increased PV+, NPY+ and GABA+ neurons in the

SupraG cell layer. This response was accompanied by a notable upregulation of cell proliferation in

both DG blades and by an increase in the number of DCX+ neurons. On the contrary, the cocaine-

treated mice exposed to behavior presented an alteration of this process. They did not upregulate their

PV+ and NPY+ populations, and their proliferating response was blunted in the InfraG blade, where

their DCX+ neurons showed abnormal morphological features suggesting a block or delay in

maturation. An insufficient or aberrant neuroadaptive response of the DG to environmental stimuli may

contribute to the cognitive symptoms induced by cocaine withdrawal. Both the hippocampal PV+ and

NPY+ interneurons and AHN have a well-established role in the hippocampal-dependent emotional

and cognitive processes (Beck and Pourie 2013; Castilla-Ortega et al. 2011; Christiansen et al. 2014;

Deng et al. 2010; Fuchs et al. 2007; Korotkova et al. 2010; Leuner et al. 2006; Morellini et al. 2010;

Quarta et al. 2015; Zou et al. 2016) and, specifically, the experience-dependent remodeling of the

hippocampal PV-related networks (Donato et al. 2013) and AHN (Castilla-Ortega et al. 2011; Deng et

al. 2010; Leuner et al. 2006) are key neuroplastic processes for memory consolidation, retrieval and

learning.

Cocaine exposure and withdrawal may notably modulate hippocampal activity and neuroplasticity

since the hippocampus receives dopaminergic projections from a main reward centre such as the

ventral tegmental area (Castilla-Ortega et al., 2016b). Acute cocaine exposure enhances hippocampal

early immediate gene expression (Schopf et al., 2015) and fMRI signal (Febo et al., 2005) but this

response attenuates after repeated dosage, which indicates synaptic changes (Febo et al., 2005). The

sculpting of the hippocampal circuitry by cocaine may involve a number of mechanisms. Cocaine

promotes the formation of dendritic spines in hippocampal neurons (Fole et al., 2011; Miguens et al.,

2015), enhances hippocampal long-term potentiation (Gabach et al., 2013; Keralapurath et al., 2014)

–which, in turn, seems occluded after long cocaine access or extended cocaine-withdrawal

(Keralapurath et al., 2015; Thompson et al., 2004)-, dysregulates hippocampal neurotransmission [e.g.

glutamate, GABA or cannabinoid signaling (Blanco et al., 2012; 2016; Li et al., 2003)], hampers

neurotrophic and inflammatory factors (Zhu et al., 2016), and temporally reduces AHN (Castilla-Ortega

et al., 2016b). Accordingly, cocaine addicts show profound neurobiological alterations in the

hippocampus, as evidenced by in vivo studies of hippocampal functional activity and connectivity [both

in basal/resting conditions and after stimulation (Adinoff et al., 2015; Castilla-Ortega et al., 2016b; Ding

and Lee, 2013)], and by post mortem gene expression analysis (Enoch et al., 2014; Enoch et al.,

2012; Mash et al., 2007; Zhou et al., 2011). Because the hippocampus reciprocally projects in the

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reward areas, it is part of the ‘cocaine addiction circuit’ (Castilla-Ortega et al., 2016b), where an

altered hippocampal activity by cocaine exposure contributes, in turn, to maintain cocaine-related

behaviors. In this regard, as revealed by clinical and/or pre-clinical experiments, the hippocampus is

involved in the acquisition and engrained retention of drug-contextual associations (Fuchs et al., 2005;

Hernandez-Rabaza et al., 2008; Otis et al., 2014), the sensitization to the stimulant locomotor effects

of cocaine (Blanco et al., 2016) –which was evidenced in our cocaine-withdrawn mice when tested in

the CPP-, and the craving and relapse outcomes elicited by cocaine-associated stimuli (Kilts et al.,

2004; Potenza et al., 2012; Tomasi et al., 2015). Although this aspect has been less explored, the fact

that both cocaine addicts and cocaine-withdrawn rodents fail in cognitive tasks that typically involve

the hippocampus [e.g. in addicts: (Aharonovich et al., 2006; Fox et al., 2009; Vonmoos et al., 2013;

Vonmoos et al., 2014); in rodents: (Briand et al., 2008; Burke et al., 2006; Krueger et al., 2009;

Mendez et al., 2008) -and the present work-] supports the idea that an aberrant hippocampal function

also contributes to the cocaine-induced cognitive decline.

Profound cognitive deficits involving global cognitive impairment are present in approximately 30 % of

cocaine addicts (and even in 12 % of cocaine recreational users) and correlate with the amount of

cocaine consumed (Vonmoos et al., 2013; 2014). During cocaine abstinence, cognitive damage may

be recovered within a year (i.e. the involved neuroadaptations seem reversed or compensated) but

only in those patients that completely cease from cocaine usage (Vonmoos et al., 2014). Furthermore,

the presence of cognitive dysfunction in cocaine addicts is a strong predictor of relapse during the first

months of drug withdrawal (Aharonovich et al., 2006; Fox et al., 2009; Teichner et al., 2002),

supporting the importance of assessing and alleviating cognitive decline in cocaine addiction. This pre-

clinical study shows that long-lasting cognitive deficits in mice withdrawn from cocaine are concomitant

to (and, probably, at least partially explained by) hippocampal alterations involving increased DG

neuronal activity, and an abnormal neuroplastic response of the interneuron populations and AHN to

environmental demands (behavioral stimulation). These results emphasize the role of the

hippocampus, and how this responds to external stimuli, as a key brain area to understand the long-

term cocaine withdrawal manifestations. Future pre-clinical research should manipulate hippocampal

inhibitory mechanisms and/or AHN to search for therapeutic approaches to treat the cocaine-induced

cognitive symptoms, and to strengthen the link between these hippocampal processes and the

behavioral effects of cocaine.

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Financial and competing interests

No competing interests declared.

Author contributions

M.M-L., M.A-D, E.C-O and L.J.S designed the experiments.

D.L.G-M, C.M, C.R-V, M.P-F., M.M., A.S., F.J.P, M.M-L. and E.C-O. performed the experiments and

contributed to data collection, analysis and/or interpretation.

M.A-D, E.C-O and L.J.S wrote the manuscript, which was revised by F.R.F. for important intellectual

content.

Funding

This research was funded by the Spanish Ministry of Economy and Competitiveness and the

European Research Development Fund (UE-ERDF) (PSI2015-73156-JIN to E.C-O, SAF12-36853 to

M. A-D, and PSI2013-44901-P to L.J.S.), Junta de Andalucía (CTS-2563 to M.A-D), and Red de

Trastornos Adictivos (RD12/0028/0001 to F.R.F.). Authors received funds from the National System of

Health: Instituto de Salud Carlos-III (Sara Borrell grant to E.C-O; code CD12/00455; and Miguel Servet

grants to A.S.; code: CP14/00173; and to F.J.P.; code: CP14/00212), from the Spanish Ministry of

Education, Culture and Sports (FPU grant to D.L.G-M.; code FPU13/04819) and from the University of

Málaga (Plan Propio grant to C.M. and C.R-V.). The funders had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript.

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Acknowledgements

We are grateful to Jody de Jong, Chiara Marcanio and Juan Gómez Repiso for their technical

assistance, to the CABIMER MR3 group (‘Retinal degeneration’) for kindly providing the experimental

room used for the behavioral assessment, and to Dr. Guillermo Estivill-Torrús for supplying the GABA

antibody employed in this study. Authors acknowledge the CABIMER and IBIMA animal facilities

(common support structure for research of Animal Experimentation; University of Málaga) for

maintenance of mice; and the IBIMA’s common support structure of Image for the use of the

microscope.

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Figures

Figure 1. Experimental protocol. Dotted lines indicate the periods where mice remained undisturbed

in their home cages. ‘Hab.’ = habituation session.

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Figure 2. Cocaine-withdrawn mice showed minimal emotional behavior alteration and no

effects on exploratory activity. The cocaine-treated mice showed a reduced number of open arm

entries in the elevated plus maze but no significant differences in the total time in open arms (a). The

COC-Behav mice performed normally in the anxiety-like measures evaluated in the light/dark box (b)

and in the open field (c) tests, as well as in the forced swimming test for depression-like behavior (d).

Locomotion, when evaluated, was unaltered (a, c). Results are represented as means ± SEM.

Student’s t tests for VEH vs COC comparisons: *p < 0.05.

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Figure 3. Cocaine withdrawal induces a persistent impairment in hippocampal-dependent

memory and sensitization to cocaine’s hyperlocomotor effects. The cocaine-withdrawn mice

showed a normal object exploration interval during the recognition sessions (a) but were unable to

remember and discriminate the novel or the displaced objects (b). In the cocaine-induced CPP

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paradigm, the COC-Behav mice showed increased hyperlocomotion when exposed to 10 mg/kg of

cocaine, but not in the saline-paired sessions or when challenged with the 2.5 mg/kg cocaine dose,

that induced no apparent stimulatory response (HAB = ‘habituation session’) (c). Conditioning was

blunted in the COC-Behav mice (d). The COC-Behav mice were also impaired in their Y maze

spontaneous alternation in absence of locomotor deficits (e). A factorial analysis that included all

memory measures confirmed a worse cognitive performance in the COC-Behav mice (f). Results are

represented as means ± SEM.

Student’s t tests for VEH vs COC comparisons: *p < 0.05, **p < 0.001.

One sample student’s t tests were used to compare means vs zero (b, d) or vs 0.5 (e): $p < 0.05 p <

0.001. [in (b), this comparison indicates a preference for the novel or the displaced object vs the

familiar or the static one; in (d) it indicates a preference for the cocaine-paired compartment vs the

saline-paired one; in (e) it indicates a frequency of spontaneous alternation over chance performance].

ANOVA ‘treatment’ effect: #p < 0.05.

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Figure 4. Increased basal c-Fos expression in the DG of mice withdrawn from cocaine. The

cocaine-withdrawn mice showed increased basal c-Fos activity in the SupraG cell layer of the DG,

irrespectively of their –Control or -Behav condition (a,b). Increased basal c-Fos activity in the DG was

also found in the –Basal mice that were not re-exposed to cocaine nor submitted to behavior (c).

Results are represented as the mean number of positive cells per mm2 ± SEM.

Post-hoc LSD test: *p < 0.05, **p < 0.001 for VEH vs COC.

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Figure 5. Chronic cocaine withdrawal dysregulates GABAergic neuron populations in the DG.

All groups of cocaine-withdrawn mice showed increased GABA+ cells in the SupraDG (a,d; and –

Basal mice in Supplementary Figure 1). Regarding the populations of PV+ and NPY+ neurons, the

COC- and VEH-treated mice showed no differences when they were not submitted to behavioral

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stimulation (b,c,e,f; Supplementary Figure 1). However, after a behavioral training, the cocaine-

withdrawn mice (COC-Behav) showed a reduced number of PV+ and NPY+ neurons in the SupraDG

compared to their VEH-Behav counterparts (b,c,e,f).

Results are represented as the mean number of positive cells per mm2 ± SEM. Arrows point positive

cells. Scales in (e) are valid for (d) and (f).

Post-hoc LSD test: *p < 0.05 for VEH vs COC.

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Figure 6. Effect of chronic cocaine withdrawal on adult hippocampal neurogenesis. The VEH-

and COC- mice showed no differences in AHN-related parameters when evaluated in –Control

conditions (a-e; Supplementary Figure 1). However, after behavioral training the COC-Behav mice

showed a reduced PCNA expression in the InfraG blade (a,d) and a reduced percent of mature-like

‘Type 2’ DCX+ neurons (c,e) in this region. Results are represented as the mean number of positive

cells per mm2 ± SEM (a,b) or as % ± SEM (c). Arrows point positive cells. In (e), the white arrows

point DCX+ ‘Type 2’ mature-like neurons, whereas black arrows point DCX+ immature-like‘Type 1’

neurons. Scales in (d) are valid for (e).

Post-hoc LSD test: *p < 0.05 for VEH vs COC.

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Figure 7. Behavior-induced modulation of plasticity in the DG is altered in cocaine-withdrawn

mice.

The graphs show the change (Δ) in DG’s marker expression induced by behavior in VEH-Behav and

COCA-Behav mice compared to their respective -Control group (represented as the zero value in the

graphs). The VEH-Behav mice showed notable behavior-induced changes, reducing basal c-Fos

expression (a) but increasing GABAergic neurons populations (b, c, d) and adult hippocampal

neurogenesis (e, f) in the DG after behavior. However, the behavior-induced upregulation of PV and

NPY was blunted in the SupraG layer of the COC-Behav mice (b, c). In addition, the COC-Behav mice

showed a blunted regulation of PCNA (e) and a reduced maturation of the young DCX+ neurons in the

InfraG blade (g).

Results are represented as difference from the respective –Control group ± SEM

Post-hoc LSD test: *p < 0.05 for VEH vs COC.

One sample student’s t tests to compare means vs zero: $p < 0.05 $$p < 0.001 (this comparison

indicates a significant change from the respective -Control group).

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Disease Models & Mechanisms 10: doi:10.1242/dmm.026682: Supplementary information

Supplementary material David Ladrón de Guevara-Miranda et al. Long-lasting memory deficits in mice withdrawn from cocaine are concomitant to neuroadaptations in hippocampal basal activity, GABAergic interneurons and adult neurogenesis *Correspondence to: manuel.alvarez@cabimer.es; estela.castilla@ibima.eu; luis@uma.es Supplementary methods for the behavioral assessment Behavioral assessment in the VEH-Behav and COC-Behav groups started at 9:00 a.m. and was performed in a noise-isolated room. Sessions were recorded and spatio-temporal parameters were analyzed with the software Ethovision XT. 7.1. (Noldus, Wageningen, The Netherlands). Object exploration and defensive behaviors in the forced swimming tests were registered by an experimented observer using Ethovision’s Manual Score module. Mice were habituated to the room for at least 20 min before the assessment began. After each session, the apparatuses were carefully cleaned with a solution of 70 % ethanol to remove the odor cues. Elevated plus maze Elevated plus maze apparatus was raised 55 cm above the floor and consisted of two open arms (37 x 5 cm) and two closed arms (37 x 5 cm surrounded by opaque walls 16.5 cm high) connected by a central platform (5 x 5 cm). Mice were placed in the center of the apparatus and allowed to explore for 5 min. Locomotion (cm), time spent in the open arms (s) and the frequency of open arm entries were analyzed. Light/dark box Light/dark box consisted of two equally sized compartments (24 x 24 cm; 23 cm high) connected by a central door. The bright compartment was painted in white color and exposed to the light in the room (~200 lux), while the dark compartment was painted in black color and covered with a lid to avoid light entry. Mice were placed in the bright compartment, facing opposite to the door, and allowed to freely explore the whole apparatus for 5 min. The total time spent in the bright field, and total number of entries to it were recorded. Open field Mice were allowed to explore a squared open field (42 x 42 cm; 40 cm high) for 5 min, to assess total locomotion, time in the center area of the maze (considered as a central 20 x 20 cm square) and number of entries in the center. Object and place recognition memory The sample trial took place three hours after the open field exploration session. Mice were again exposed to the same open field apparatus but containing two identical copies of one object (‘familiar’ object) located in two adjacent corners. On the following day (24 hours interval), the objects were replaced by another copy of the familiar object and by an unknown object (‘novel’ object) located in the same previous positions. Finally, 24 hours later, mice faced again two copies of the familiar object, one was placed in its previous position (‘static’ object), but the other was displaced to a novel position in the opposite corner of the maze (‘displaced’ object) (an scheme of this setting is depicted in Fig. 3a, in the main manuscript). The duration of the sessions was 10 min. The objects used for this task were made from laboratory expendables with different shapes (a 50 ml conical tube and a 50 ml cell culture flask) that were covered with material from a nitrile glove and/or adhesive tape so they differed in both color and texture. Both the positions of the objects in the maze and the type of object designated as ‘familiar’ or ‘novel’ were counterbalanced across mice. The total time of object exploration (defined as the mouse touching an object with its nose or forepaws) was scored, and both object and place recognition memories were calculated by

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Disease Models & Mechanisms 10: doi:10.1242/dmm.026682: Supplementary information

the following discrimination ratios: ‘Object memory ratio’ = [(time exploring the novel object – time exploring the familiar object)/ total time exploring both objects]; ‘Place memory ratio’ = [(time exploring the displaced object – time exploring the static object)/ total time exploring both objects] (Albasser et al. 2010; Barker and Warburton 2011). The resulting ratio can vary between +1 and −1, so a preference for the novel (vs familiar) or for the displaced (vs static) object would yield a positive ratio score significantly different from zero; while a score equal to zero would indicate absence of discrimination (Albasser et al. 2010). Forced swimming test Depression-like behavior was evaluated in a 5 min session in the forced swimming test. Mice were placed in a clear cylinder (27 cm high, 10 cm diameter) filled with water (22 ± 1°C) to a height of 15 cm. The total time of immobility (the mouse floats passively, making only those movements necessary to keep its head above the water), the latency to the first immobility and the total time of struggling behavior (the mouse is highly active, trying to escape by climbing the cylinder’s walls) were recorded. Cocaine-induced conditioned place preference The cocaine-induced conditioned place preference (CPP) protocol was based on previously published methods (Castilla-Ortega et al. 2016; Ladron de Guevara-Miranda et al. 2016). (Fig. 1a, in the main manuscript). The CPP apparatus (Panlab, Barcelona, Spain) consisted of two equally sized compartments (20 x 18 cm, 25 cm height) interconnected by a corridor made of clear plexiglass. The compartments were different from each other in the pattern decorating their walls (white dots over a black background vs stripes in white and gray tones) and in the shape of their corners. Mice first received one habituation session (CPP day 1) where they freely explored the entire apparatus for 15 min, and the time spent in each compartment was recorded to assess the spontaneous preference. The conditioning training was then performed in two consecutive phases using increasing cocaine doses: 2.5 mg/kg (CPP days 2-5) and 10 mg/kg (CPP days 7-10). Each conditioning phase included four training days in which mice received one cocaine- and one saline-paired session, each session consisting of an i.p. administration of cocaine or saline followed by immediate confinement in one compartment for 15 min. For mice that preferred one compartment in the habituation session (difference in time > 15%), the compartment paired with cocaine was the least preferred one. The daily order of the cocaine- and saline-paired sessions was counterbalanced across days (1st day: cocaine/saline; 2nd: saline/cocaine; 3th: cocaine/saline; 4th: saline/cocaine) and sessions were separated for at least 4 hours. A Test session was carried out after each conditioning phase (CPP days 6 and 11), in which mice were administered saline and explored the entire apparatus for 15 min in order to assess place preference. A ‘CPP ratio’ [(seconds spent in the cocaine-paired compartment - seconds spent in the saline-paired compartment) / total seconds spent in both compartments]*100 was calculated for the habituation and tests sessions. The preference for the cocaine-paired compartment over the saline-paired one would be indicated by a positive CPP ratio score, significantly different from zero. Locomotion was assessed through the entire test. Y Maze The Y maze consisted of three equal arms (40 x 9 cm, 16 cm high; named A, B, C) interconnected at 120 degree angles. The mouse was placed in one arm and allowed to explore freely. The sequence of arm entries (e.g. ACBACA) was registered to assess the total number of spontaneous alternations. One spontaneous alternation was defined as three successive entries in different arms (e.g. ‘ACB’, ‘CBA’, ‘BAC’ but not ‘ACA’) (Hughes 2004). An ‘alternation score’ was calculated = [(number of spontaneous alternations)/(total the number of arm entries – 2)]. Total locomotion was analyzed across 5 min, while the alternation score was analyzed until mice performed 32 arm entries (i.e. 30 possible alternations).

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Disease Models & Mechanisms 10: doi:10.1242/dmm.026682: Supplementary information

Supplementary methods for the statistical analysis Principal Components Factor Analysis Following our previous methods (Castilla-Ortega et al. 2016), a principal components factorial analysis (PCA) with varimax rotation was performed on the memory-related measures (i.e. the object memory ratio, the place memory ratio, the CPP ratio in each test session and the Y-maze alternation score) to test whether they would share the same behavioural dimension. Importantly, data achieved the criterion for sample adequacy to perform a PCA (Kaiser-Meyer-Olkin index = 0.649; Bartlett’s test for sphericity: X2 = 19.995, df = 10, p = 0.029). Relevant factors were selected using the criterion of the eigenvalue ≥ 1, and a behaviour was considered as included in a factor when its contribution (i.e., ‘factor loading’) was ≥ 0.5 in absolute value. After extracting the factor, the score for each mice in the behavioral dimension (i.e. Factor Score) was calculated by the regression method. Behavior-induced modulation of histological markers These analysis tested whether the magnitude of the neuroplastic change induced by behavior was significantly different between the VEH-Behav and the COC-Behav mice. Data of mice in the –Behav groups were transformed to a ‘change score’ indicating the relative change from their control group (VEH-Control or COC-Control). This was calculated for each histological marker by subtracting the mean value of the cells quantified in the respective control group from the number of cells quantified in each –Behav mice. Thus, a negative change score would indicate that the number of cells was reduced by behavior; a positive score would indicate increased expression; and a change score near zero would indicate that behavior had no effects.

References:

Albasser MM, Chapman RJ, Amin E, Iordanova MD, Vann SD, Aggleton JP (2010) New behavioral protocols to extend our knowledge of rodent object recognition memory. Learning & memory (Cold Spring Harbor, NY) 17 (8):407-419. doi:10.1101/lm.1879610

Barker GR, Warburton EC (2011) When is the hippocampus involved in recognition memory? The Journal of neuroscience : the official journal of the Society for Neuroscience 31 (29):10721-10731. doi:10.1523/jneurosci.6413-10.2011

Castilla-Ortega E, Blanco E, Serrano A, Ladron de Guevara-Miranda D, Pedraz M, Estivill-Torrus G, Pavon FJ, Rodriguez de Fonseca F, Santin LJ (2016) Pharmacological reduction of adult hippocampal neurogenesis modifies functional brain circuits in mice exposed to a cocaine conditioned place preference paradigm. Addiction biology 21 (3):575-588. doi:10.1111/adb.12248

Hughes RN (2004) The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory. Neuroscience and biobehavioral reviews 28 (5):497-505. doi:10.1016/j.neubiorev.2004.06.006

Ladron de Guevara-Miranda D, Pavon FJ, Serrano A, Rivera P, Estivill-Torrus G, Suarez J, Rodriguez de Fonseca F, Santin LJ, Castilla-Ortega E (2016) Cocaine-conditioned place preference is predicted by previous anxiety-like behavior and is related to an increased number of neurons in the basolateral amygdala. Behavioural brain research 298 (Pt B):35-43. doi:10.1016/j.bbr.2015.10.048

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Disease Models & Mechanisms 10: doi:10.1242/dmm.026682: Supplementary information

Supplementary Figure 1

  

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SFigure 1: Histological results in the –Basal mice for interneurons and adult neurogenesis-related markers. Compared to the VEH-Basal mice, the COC–Basal mice (protocol described in Fig. 1c in the main manuscript) showed increased GABA+ neurons in the SupraG cell layer (‘treatment’: F(1, 10) = 7.666, p = 0.020; ‘DG Blade’: F(1, 10) = 65.196, p = 0.000; similar to the COC-treated groups reported in Fig. 5a,d, in the main manuscript) but they did not show further significant differences in the PV and NPY populations or in the AHN-related markers. Post-hoc LSD test: *p < 0.05 for –VEH vs –COC.

VEH-BasalCOC-Basal*

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Disease Models & Mechanisms 10: doi:10.1242/dmm.026682: Supplementary information

Supplementary Figure 2

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  SFigure 2: Apoptotic nuclei in the DG in the –Behav mice. Apoptosis determination was performed with an in situ apoptosis detection kit (NeuroTACS-II, Trevigen, Gaithersburg, USA) following the manufacturer's instructions. Apoptotic nuclei were quantified in the –Behav mice in one representative section per animal selected approximately at bregma -2.06 mm. Apoptosis data suggest a normal apoptotic death of the adult-born neurons in the COC-Behav mice, since no differences were found between groups and the apoptosis graph resembled the cell proliferation data described for these animals (Fig. 6a in the main manuscript). Apoptotic nuclei quantified specifically in the subgranular zone (SGZ) did not reveal differences either (data not shown).

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Disease Models & Mechanisms 10: doi:10.1242/dmm.026682: Supplementary information

Supplementary Table

C-Fos expression and GABAergic populations in the hippocampus of the VEH-Behav and the COC-Behav mice. Groups were compared by a repeated measures ANOVA (‘treatment’ x ‘stratum’) carried out independently in the CA1, CA3 or DG regions. Results only revealed a reduction of the NPY+ neurons in the CA1-oriens of the COC-Behav mice (‘stratum’: F(3, 57) = 177.59, p = 0.000; ‘stratum x treatment’: F(3, 57) = 10.457, p = 0.000; LSD is shown in the table).

c-Fos+ GABA+ PV+ NPY

VEH-Behav COC-Behav VEH-Behav COC-Behav VEH-Behav COC-Behav VEH-Behav COC-Behav

CA1

Oriens 34.19 ± 2.36 34.25 ± 3.91 10.29 ± 1.02 10.15 ± 0.93 18.36* ± 1.22 13.84* ± 0.64

Pyramidal 167.36 ±

11.88 157.78 ±

13.06 27.25 ± 1.26 27.76 ± 2.55 20.81 ± 1.96 22.12 ± 1.95 9.35 ± 1.81 9.88 ± 0.89

Radiatum 19.72 ± 1.09 20.64 ± 1.65 1.46 ± 0.15 2.26 ± 0.27 4.29 ± 0.30 4.31 ± 0.39

Lacunosum

84.13 ± 6.20 80.85 ± 4.33 - - 6.70 ± 0.40 6.63 ± 0.40

CA3

Oriens 46.15 ± 6.58 41.85 ± 8.03 16.65 ± 1.60 16.75 ± 1.43 19.23 ± 2.03 19.02 ± 3.14

Pyramidal 133.41 ±

13.63 140.67 ±

10.27 28.76 ± 3.11 25.22 ± 2.46 22.20 ± 1.81 23.00 ± 2.12 5.00 ± 0.71 4.72 ± 0.26

Radiatum 63.38 ± 6.51 61.02 ± 6.02 7.89 ± 0.62 7.02 ± 0.69 12.69 ± 1.17 12.69 ± 1.17

DG Molecular 6.04 ± 0.29 7.14 ± 0.73 0.44 ± 0.10 0.36 ± 0.05 1.77 ± 0.24 1.65 ± 0.22

Polymorphic 13.39 ± 3.11 17.14 ± 2.75 2.99 ± 0.39 3.87 ± 0.52 41.49 ± 3.20 40.81 ± 3.99

Results are expressed as mean positive cells per mm2 ± SEM *p < 0.001 (post-hoc LSD vs the other group)

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